@article {69755, title = {Quantitative Profiling pH Heterogeneity of Acidic Endolysosomal Compartments using Fluorescence Lifetime Imaging Microscopy.}, journal = {Mol Biol Cell}, year = {2025}, month = {2025 Jan 29}, pages = {mbcE23060220}, abstract = {

The endo-lysosomal system plays a crucial role in maintaining cellular homeostasis and promoting organism fitness. The pH of its acidic compartments is a crucial parameter for proper function, and it is dynamically influenced by both intracellular and environmental factors. Here, we present a method based on fluorescence lifetime imaging microscopy (FLIM) for quantitatively analyzing the pH profiles of acidic endolysosomal compartments in diverse types of primary mammalian cells and in live organism . This FLIM-based method exhibits high sensitivity in resolving subtle pH differences, thereby revealing heterogeneity within a cell and across cell types. This method enables rapid measurement of pH changes in the acidic endolysosomal system in response to various environmental stimuli. Furthermore, the fast FLIM measurement of pH-sensitive dyes circumvents the need for transgenic reporters and mitigates potential confounding factors associated with varying dye concentrations or excitation light intensity. This FLIM approach offers absolute pH quantification and highlights the significance of pH heterogeneity and dynamics, offering a valuable tool for investigating lysosomal functions and their regulation in various physiological and pathological contexts.

}, issn = {1939-4586}, doi = {10.1091/mbc.E23-06-0220}, author = {Deng, Dinghuan and Guan, Youchen and Mutlu, Ayse Sena and Wang, Baiping and Gao, Shihong Max and Zheng, Hui and Wang, Meng C} } @article {6956, title = {Structural basis for the prion-like MAVS filaments in antiviral innate immunity.}, journal = {eLife}, volume = {3}, year = {2014}, month = {2014 Jan 1}, pages = {e01489}, abstract = {

Mitochondrial antiviral signaling (MAVS) protein is required for innate immune responses against RNA viruses. In virus-infected cells MAVS forms prion-like aggregates to activate antiviral signaling cascades, but the underlying structural mechanism is unknown. Here we report cryo-electron microscopic structures of the helical filaments formed by both the N-terminal caspase activation and recruitment domain (CARD) of MAVS and a truncated MAVS lacking part of the proline-rich region and the C-terminal transmembrane domain. Both structures are left-handed three-stranded helical filaments, revealing specific interfaces between individual CARD subunits that are dictated by electrostatic interactions between neighboring strands and hydrophobic interactions within each strand. Point mutations at multiple locations of these two interfaces impaired filament formation and antiviral signaling. Super-resolution imaging of virus-infected cells revealed rod-shaped MAVS clusters on mitochondria. These results elucidate the structural mechanism of MAVS polymerization, and explain how an α-helical domain uses distinct chemical interactions to form self-perpetuating filaments. DOI: http://dx.doi.org/10.7554/eLife.01489.001.

}, issn = {2050-084X}, doi = {10.7554/eLife.01489}, author = {Xu, Hui and He, Xiaojing and Zheng, Hui and Huang, Lily J and Hou, Fajian and Yu, Zhiheng and de la Cruz, Michael Jason and Borkowski, Brian and Zhang, Xuewu and Chen, Zhijian J and Jiang, Qiu-Xing} } @article {65045, title = {Voltage sensor ring in a native structure of a membrane-embedded potassium channel.}, journal = {Proceedings of the National Academy of Sciences of the United States of America}, volume = {110}, year = {2013}, month = {2013 Feb 26}, pages = {3369-74}, abstract = {

Voltage-gated ion channels support electrochemical activity in cells and are largely responsible for information flow throughout the nervous systems. The voltage sensor domains in these channels sense changes in transmembrane potential and control ion flux across membranes. The X-ray structures of a few voltage-gated ion channels in detergents have been determined and have revealed clear structural variations among their respective voltage sensor domains. More recent studies demonstrated that lipids around a voltage-gated channel could directly alter its conformational state in membrane. Because of these disparities, the structural basis for voltage sensing in native membranes remains elusive. Here, through electron-crystallographic analysis of membrane-embedded proteins, we present the detailed view of a voltage-gated potassium channel in its inactivated state. Contrary to all known structures of voltage-gated ion channels in detergents, our data revealed a unique conformation in which the four voltage sensor domains of a voltage-gated potassium channel from Aeropyrum pernix (KvAP) form a ring structure that completely surrounds the pore domain of the channel. Such a structure is named the voltage sensor ring. Our biochemical and electrophysiological studies support that the voltage sensor ring represents a physiological conformation. These data together suggest that lipids exert strong effects on the channel structure and that these effects may be changed upon membrane disruption. Our results have wide implications for lipid-protein interactions in general and for the mechanism of voltage sensing in particular.

}, keywords = {Aeropyrum, Cell Membrane, Cross-Linking Reagents, Crystallography, X-Ray, Electrons, Lipids, Models, Molecular, Potassium Channels, Voltage-Gated, Protein Structure, Secondary, Protein Structure, Tertiary}, issn = {1091-6490}, doi = {10.1073/pnas.1218203110}, author = {Shi, Liang and Zheng, Hongjin and Zheng, Hui and Borkowski, Brian A and Shi, Dan and Gonen, Tamir and Jiang, Qiu-Xing} }